Method and device for detecting ammonia odors and helicobacter pylori urease infection Patent Grant Boga , et al. September 1, 2 [Kimberly-Clark Worldride, Inc.]

Method and device for detecting ammonia odors and helicobacter pylori urease infection

Boga , et al. September 1, 2

Patent Grant 7582485

U.S. patent number 7,582,485 [Application Number 10/687,327] was granted by the patent office on 2009-09-01 for method and device for detecting ammonia odors and helicobacter pylori urease infection. This patent grant is currently assigned to Kimberly-Clark Worldride, Inc.. Invention is credited to RameshBabu Boga, John Gavin MacDonald.

United States Patent	7,582,485
Boga , et al.	September 1, 2009

**Please see images for: ( Certificate of Correction ) **

Method and device for detecting ammonia odors and helicobacter pylori urease infection

Abstract

The invention provides a breath testing device which visually indicates the presence of ammonia in a patient's breath, in particular ammonia from helicobacter pylori urease infection. The breath testing device comprises a visual indicating agent which changes color in response to ammonia odors, such as 4,4'-bis(dimethylamino)-benzhydrol (Michler's hydrol or BDMB), pararosaniline base and alpha-naphtholbenzein. The indicating agent is applied to a substrate which is then inserted into a tube or straw, which can be attached to the inlet of a breath collection balloon. When the patient blows into the tube or straw, the indicating agent will change color if it detects levels of ammonia which are consistent with helicobacter pylori urease infection.

Inventors:	Boga; RameshBabu (Roswell, GA), MacDonald; John Gavin (Decatur, GA)
Assignee:	Kimberly-Clark Worldride, Inc. (Neenah, WI)
Family ID:	34520944
Appl. No.:	10/687,327
Filed:	October 16, 2003

Prior Publication Data


	Document Identifier	Publication Date
	US 20050084977 A1	Apr 21, 2005

Current U.S. Class:	436/113; 422/413; 435/12; 435/7.1; 600/532
Current CPC Class:	G01N 31/223 (20130101); G01N 33/497 (20130101); G01N 33/56922 (20130101); Y10T 436/175383 (20150115)
Current International Class:	G01N 33/53 (20060101); G01N 33/00 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


2015864	October 1935	Muller et al.
2429694	October 1947	King
2534229	December 1950	Carhart et. al.
2593146	April 1952	Howard
3033655	May 1962	Grosskopf
3193404	July 1965	Davis
3266973	August 1966	Crowley
3338992	August 1967	Kinney
3341394	September 1967	Kinney
3381688	May 1968	Satas
3494821	February 1970	Evans
3502538	March 1970	Petersen
3502763	March 1970	Hartmann
3507269	April 1970	Berry
3542615	November 1970	Dobo et al.
3615478	October 1971	Hoshino et al.
3692618	September 1972	Dorschner et al.
3794497	February 1974	Pratt et al.
3802817	April 1974	Matsuki et al.
3836633	September 1974	Bescke
3842103	October 1974	Smith
3849241	November 1974	Butin et al.
3919437	November 1975	Brown et al.
3960494	June 1976	Verma et al.
3971665	July 1976	Suzuki et al.
4006030	February 1977	Yoshida et al.
4038101	July 1977	Thompson
4041203	August 1977	Brock et al.
4078029	March 1978	Yoshida et al.
4100324	July 1978	Anderson et al.
4101638	July 1978	Inoue et al.
4144370	March 1979	Boulton
4172781	October 1979	Walk et al.
4243743	January 1981	Hiramoto et al.
4297233	October 1981	Gualandi
RE30797	November 1981	Davis
RE30803	November 1981	Davis
4313820	February 1982	Farha, Jr. et al.
4340563	July 1982	Appel et al.
4375448	March 1983	Appel et al.
4407960	October 1983	Tratnyek
4467012	August 1984	Pedersen et al.
4469746	September 1984	Weisman et al.
4488969	December 1984	Hou
4494278	January 1985	Kroyer et al.
4494629	January 1985	Raeburn
4517308	May 1985	Ehlenz et al.
4522203	June 1985	Mays
4525410	June 1985	Hagiwara et al.
4548906	October 1985	Sekikawa et al.
4575556	March 1986	Byrne et al.
4604313	August 1986	McFarland et al.
4640810	February 1987	Laursen et al.
4643801	February 1987	Johnson
4655757	April 1987	McFarland et al.
4670385	June 1987	Babb et al.
4701218	October 1987	Barker et al.
4715983	December 1987	Ota et al.
4725415	February 1988	Kidd
4734324	March 1988	Hill
RE32649	April 1988	Brandt et al.
4769216	September 1988	Chandler et al.
4775585	October 1988	Hagiwara et al.
4777018	October 1988	Auchincloss
4780448	October 1988	Broecker et al.
4781858	November 1988	Mizukami et al.
4783220	November 1988	Gamble et al.
4798603	January 1989	Meyer et al.
4802473	February 1989	Hubbard et al.
4804630	February 1989	Bhattacharjee
4818464	April 1989	Lau
4823404	April 1989	Morell et al.
4823803	April 1989	Nakamura
4844867	July 1989	Bather
4853186	August 1989	Mura et al.
4854332	August 1989	Hanakura
4904304	February 1990	Watanabe et al.
4947861	August 1990	Hamilton
4969457	November 1990	Hubbard et al.
4978615	December 1990	Aoyama et al.
4988505	January 1991	Watanabe et al.
5000746	March 1991	Meiss
5020533	June 1991	Hubbard et al.
5057302	October 1991	Johnson et al.
5064473	November 1991	Kubo et al.
5094962	March 1992	Snyder et al.
5100581	March 1992	Watanabe et al.
5100702	March 1992	Maeda et al.
5108739	April 1992	Kurihara et al.
5122418	June 1992	Nakane et al.
5133803	July 1992	Moffatt
5145518	September 1992	Winnik et al.
5145727	September 1992	Potts et al.
5169706	December 1992	Collier, IV et al.
5174959	December 1992	Kundu et al.
5178931	January 1993	Perkins et al.
5183656	February 1993	Uesaka et al.
5188885	February 1993	Timmons et al.
5196177	March 1993	Watanabe et al.
5204429	April 1993	Kaminsky et al.
5209998	May 1993	Kavassalis et al.
5220000	June 1993	Theodoropulos
5221497	June 1993	Watanabe et al.
5225374	July 1993	Fare et al.
5230953	July 1993	Tsugeno et al.
5238518	August 1993	Okubi et al.
5245117	September 1993	Withers et al.
5266289	November 1993	Tsugeno et al.
5284703	February 1994	Everhart et al.
5286624	February 1994	Terashima et al.
5292868	March 1994	Subramanian
5294717	March 1994	Theodoropulos
5300365	April 1994	Ogale
5322061	June 1994	Brunson
5332432	July 1994	Okubi et al.
5338713	August 1994	Takagi et al.
5342876	August 1994	Abe et al.
5350624	September 1994	Georger et al.
5366947	November 1994	Muller et al.
5382400	January 1995	Pike et al.
5383450	January 1995	Hubbard et al.
5397667	March 1995	Law et al.
5407442	April 1995	Karapasha
5407600	April 1995	Ando et al.
5420016	May 1995	Boguslaski et al.
5420090	May 1995	Spencer et al.
5427844	June 1995	Murai et al.
5429628	July 1995	Trinh et al.
5432094	July 1995	Delente
5451450	September 1995	Erderly et al.
5458864	October 1995	Tsugeno et al.
5472775	December 1995	Obijeski et al.
5480636	January 1996	Maruo et al.
5486356	January 1996	Yim
5487938	January 1996	Spencer et al.
5488126	January 1996	Subramanian et al.
5527171	June 1996	Soerensen
5529879	June 1996	Hoshino et al.
5538548	July 1996	Yamazaki
5539124	July 1996	Etherton et al.
5540916	July 1996	Parks
5547607	August 1996	Ando et al.
5553608	September 1996	Reese et al.
5554775	September 1996	Krishnamurti et al.
5580655	December 1996	El-Shall et al.
5583219	December 1996	Subramanian et al.
5591797	January 1997	Barthel et al.
5597512	January 1997	Watanabe et al.
5661198	August 1997	Inatani et al.
5663224	September 1997	Emmons et al.
5679138	October 1997	Bishop et al.
5679724	October 1997	Sacripante et al.
5695868	December 1997	McCormack
5733272	March 1998	Brunner et al.
5773227	June 1998	Kuhn et al.
5795985	August 1998	Husler et al.
5813398	September 1998	Baird et al.
5814370	September 1998	Martino et al.
5817300	October 1998	Cook et al.
5833952	November 1998	Grigor et al.
5837352	November 1998	English et al.
5843509	December 1998	Calvo Salve et al.
5855788	January 1999	Everhart et al.
5861144	January 1999	Peterson et al.
5871872	February 1999	Matijevic et al.
5874067	February 1999	Lucas et al.
5880176	March 1999	Kamoto et al.
5880309	March 1999	Suzuki et al.
5882638	March 1999	Dodd et al.
5885599	March 1999	Peterson et al.
5897541	April 1999	Uitenbroek et al.
5902226	May 1999	Tasaki et al.
5905101	May 1999	Fujiki et al.
5916596	June 1999	Desai et al.
5948398	September 1999	Hanamoto et al.
5948483	September 1999	Kim et al.
5962566	October 1999	Grandfils et al.
5964926	October 1999	Cohen
5972389	October 1999	Shell et al.
5985229	November 1999	Yamada et al.
5989510	November 1999	Abe et al.
5989515	November 1999	Watanabe et al.
5989840	November 1999	D'Angelo et al.
6004625	December 1999	Ohshima
6007592	December 1999	Kasai et al.
6024786	February 2000	Gore
6039923	March 2000	Klemm et al.
6045900	April 2000	Haffner et al.
6047413	April 2000	Welchel et al.
6057162	May 2000	Rounbehler et al.
6060410	May 2000	Gillberg-LaForce et al.
6073771	June 2000	Pressley et al.
6075179	June 2000	McCormack et al.
6096299	August 2000	Guarracino et al.
6111163	August 2000	McCormack et al.
6171811	January 2001	Becerro De Bengoa Vallejo
6172173	January 2001	Spencer et al.
6177608	January 2001	Weinstrauch
6190814	February 2001	Law et al.
6193844	February 2001	McLaughlin et al.
6225524	May 2001	Guarracino et al.
6228605	May 2001	Marshall
6238767	May 2001	McCormack et al.
6254894	July 2001	Denkewicz, Jr. et al.
6264615	July 2001	Diamond et al.
6277346	August 2001	Murasawa et al.
6277772	August 2001	Gancet et al.
6291535	September 2001	Watanabe et al.
6294222	September 2001	Cohen et al.
6299867	October 2001	Aoyagi et al.
6309736	October 2001	McCormack et al.
6312918	November 2001	Ito et al.
6315864	November 2001	Anderson et al.
6334988	January 2002	Gallis et al.
6344218	February 2002	Dodd et al.
6344272	February 2002	Oldenburg et al.
6358537	March 2002	Hoshino et al.
6358909	March 2002	Ochomogo et al.
6369290	April 2002	Glaug et al.
6376741	April 2002	Guarracino et al.
6387495	May 2002	Reeves et al.
6391262	May 2002	Brinton et al.
6398827	June 2002	Ota et al.
6406669	June 2002	Duan et al.
6410765	June 2002	Wellinghoff et al.
6416729	July 2002	DeBerry et al.
6425530	July 2002	Coakley
6427693	August 2002	Blackstock et al.
6428814	August 2002	Bosch et al.
6433243	August 2002	Woltman et al.
6440187	August 2002	Kasai et al.
6460989	October 2002	Yano et al.
6461735	October 2002	Furuya et al.
6467897	October 2002	Wu et al.
6468500	October 2002	Sakaguchi et al.
6475601	November 2002	Sakaki et al.
6479150	November 2002	Liu et al.
6479278	November 2002	Marshall
6491790	December 2002	Proverb et al.
6498000	December 2002	Murasawa et al.
6509169	January 2003	Ratcliffe et al.
6517199	February 2003	Tomioka et al.
6531704	March 2003	Yadav et al.
6533826	March 2003	Gessner et al.
6536890	March 2003	Kato et al.
6544492	April 2003	DeBerry
6548264	April 2003	Tan et al.
6551457	April 2003	Westman et al.
6562441	May 2003	Maeda et al.
6575383	June 2003	Dobler et al.
6578521	June 2003	Raymond et al.
6589562	July 2003	Shefer et al.
6589761	July 2003	Freadman et al.
6607711	August 2003	Pedersen
6617488	September 2003	Springer et al.
6623848	September 2003	Brehm et al.
6627377	September 2003	Itatani et al.
6638918	October 2003	Davison et al.
6639004	October 2003	Falat et al.
6645569	November 2003	Cramer et al.
6693071	February 2004	Ghosh et al.
6706537	March 2004	Ignacio et al.
6825040	November 2004	Scaringe et al.
6846956	January 2005	Kast et al.
6998250	February 2006	McMichael et al.
7014612	March 2006	Hubbard et al.
7022285	April 2006	Arai et al.
7052854	May 2006	Melker et al.
7101340	September 2006	Braun
7316899	January 2008	McDevitt et al.
7413550	August 2008	MacDonald et al.
2001/0000889	May 2001	Yadav et al.
2001/0023338	September 2001	Guarracino et al.
2001/0031248	October 2001	Hall-Puzio et al.
2001/0056246	December 2001	Rodriguez-Fernandez et al.
2002/0005145	January 2002	Sherman
2002/0066542	June 2002	Jakob et al.
2002/0068364	June 2002	Arai et al.
2002/0091071	July 2002	Fischer et al.
2002/0106466	August 2002	Hausmann et al.
2002/0110686	August 2002	Dugan
2002/0128336	September 2002	Kolb et al.
2002/0142937	October 2002	Carter et al.
2002/0149656	October 2002	Nohr et al.
2002/0150678	October 2002	Cramer et al.
2002/0176982	November 2002	Rohrbaugh et al.
2002/0177621	November 2002	Hanada
2002/0182102	December 2002	Fontenot et al.
2002/0182658	December 2002	Polak et al.
2003/0003589	January 2003	Khalil et al.
2003/0013369	January 2003	Soane et al.
2003/0021744	January 2003	DeBerry et al.
2003/0021983	January 2003	Nohr et al.
2003/0050211	March 2003	Hage et al.
2003/0056648	March 2003	Fornai et al.
2003/0070782	April 2003	Proverb et al.
2003/0082237	May 2003	Cha et al.
2003/0100842	May 2003	Rosenberg et al.
2003/0130631	July 2003	Springer et al.
2003/0147956	August 2003	Shefer et al.
2003/0147966	August 2003	Franzen et al.
2003/0181540	September 2003	Quellet et al.
2003/0203009	October 2003	MacDonald
2003/0203495	October 2003	Rupp
2003/0211618	November 2003	Patel
2003/0216660	November 2003	Ben-Oren et al.
2003/0235605	December 2003	Lelah et al.
2004/0033269	February 2004	Hei et al.
2004/0034157	February 2004	Ghosh et al.
2004/0043688	March 2004	Soerens et al.
2004/0077093	April 2004	Pan
2004/0122387	June 2004	Long et al.
2004/0175556	September 2004	Clark et al.
2004/0265742	December 2004	Marck et al.
2005/0012085	January 2005	Lavi et al.
2005/0037374	February 2005	Melker et al.
2005/0084977	April 2005	Boga et al.
2005/0085739	April 2005	MacDonald et al.
2005/0112085	May 2005	MacDonald et al.
2005/0124072	June 2005	Boga et al.
2005/0164320	July 2005	McDevitt et al.
2005/0186109	August 2005	Nakamura et al.
2005/0191704	September 2005	Boga et al.
2006/0003336	January 2006	Song et al.
2007/0249958	October 2007	Martin et al.
2008/0243015	October 2008	MacDonald et al.

Foreign Patent Documents


0103214	Mar 1984	EP
0232141	Aug 1987	EP
0251783	Jan 1988	EP
0339461	Nov 1989	EP
0348978	Jan 1990	EP
0376448	Jul 1990	EP
0389015	Sep 1990	EP
0389023	Sep 1990	EP
0483500	May 1992	EP
0510619	Oct 1992	EP
0282287	Apr 1996	EP
0972563	Jan 2000	EP
0749295	Jul 2000	EP
1034800	Sep 2000	EP
001037045	Sep 2000	EP
1053788	Nov 2000	EP
1157672	Nov 2001	EP
1157672	Nov 2001	EP
001160569	Dec 2001	EP
1162172	Dec 2001	EP
1188854	Mar 2002	EP
1214878	Jun 2002	EP
1216675	Jun 2002	EP
1298071	Apr 2003	EP
1315526	Jun 2003	EP
62149322	Jul 1987	JP
3221142	Sep 1991	JP
92067913	Oct 1992	JP
WO 8902698	Apr 1989	WO
WO 9111977	Aug 1991	WO
WO 9112029	Aug 1991	WO
WO 9112030	Aug 1991	WO
WO 9619346	Jun 1996	WO
WO 9619346	Jun 1996	WO
WO 9705482	Feb 1997	WO
WO 9725076	Jul 1997	WO
97/30351	Aug 1997	WO
WO 98/20915	May 1998	WO
WO 9820915	May 1998	WO
WO 98/26808	Jun 1998	WO
WO 9826808	Jun 1998	WO
WO 9826808	Jun 1998	WO
WO 99/47252	Sep 1999	WO
WO 00/03797	Jan 2000	WO
WO 0029036	Mar 2000	WO
WO 0029036	Mar 2000	WO
WO 00137764	Mar 2000	WO
00/20852	Apr 2000	WO
WO 0059555	Oct 2000	WO
WO 00/76558	Dec 2000	WO
WO 007658	Dec 2000	WO
WO 01/06054	Jan 2001	WO
WO 02/26272	Apr 2002	WO
WO 02/49559	Jun 2002	WO
WO 02/55115	Jul 2002	WO
WO 02/062881	Aug 2002	WO
WO 02/064877	Aug 2002	WO
WO 02/083297	Oct 2002	WO
WO 02/084017	Oct 2002	WO
WO 02/095112	Nov 2002	WO
WO 02094329	Nov 2002	WO
WO 03/000979	Jan 2003	WO
WO 03/025067	Mar 2003	WO
WO 03032959	Apr 2003	WO
WO 03088931	Oct 2003	WO
WO 03/092885	Nov 2003	WO
WO 2004000986	Dec 2003	WO

Other References

Hart, Courtenay et al, Electrophoresis, vol. 24, pp. 588-598, 2003. cited by examiner .
Rottman, Claudio et al, J. Am. Chem. Soc., vol. 123, pp. 5730-5734, 2001, Getting a library of activities from a single compound: Tunability and very large shifts in acidiity constants induced by sol-gel entrapped micells. cited by examiner .
Dunn, CDR et al, British Journal of Biomedical Science, vol. 58, pp. 66-75, 2001, Ammonia vapor in the mouth as a diagnostic marker for Helicobacter pylori infection: preliminary `proof of principle` pharmacological investigations. cited by examiner .
Kearney, David J. et al, Digestive Disorders and Sciences, vol. 47(11) Nov. 2002, pp. 2523-2530, Breath Ammonia Measurement in Helicobacter pylori infection. cited by examiner .
Article --Immunization of mice with peptomers covalently couopled to aluminum oxide nanoparticles, Andreas Frey, Nicholas Mantis, Pamela A. Kozlowski, Alison J. Quayle, Adriana Bajardi, Juana J. Perdomo, Frank A. Robey, and Marian R. Neutra, Vaccine, vol. 17, 1999, pp. 3007-3019. cited by other .
PCT Search Report for PCT/US03/39737, Jun. 18, 2004. cited by other .
Abstract of Japanese Patent No. JPO4255767, Sep. 10, 1992. cited by other .
Abstract of Japanese Patent No. JP05098185, Apr. 20, 1993. cited by other .
Abstract of Japanese Patent No. JP57135360, Aug. 20, 1982. cited by other .
Abstract of Japanese Patent No. JP2001208753, Mar. 8, 2001. cited by other .
Abstract of Japanese Patent No. JP198610366, Jan. 11, 1986. cited by other .
Article--A sorbent tube for oral malodour monitoring, Julia Rodriguez-Fernandez, Regina Lopez-Fernandez, Rosario Pereiro, Manuel Menendez, JoseMaria Tejerina, Alberto Sicilia, and Alfredo Sanz-Medel, Talanta, vol. 62, 2004, pp. 421-426. cited by other .
Article--Optical fibre sensor for hydrogen sulphide monitoring in mouth air, Julio Rodriguez-Fernandez, Rosario Pereiro, and Alfredo Sanz-Medel, Analytica Chimica Acta, vol. 471, 2002, pp. 13-23. cited by other .
U.S. Appl. No. 10/723,761, filed Nov. 26, 2004, Quincy, III, et al. Odor Control In Personal Care Products. cited by other .
U.S. Appl. No. 10/955,316, filed Sep. 30, 2004, MacDonald, et al. Odor-Reducing Quinone Compounds. cited by other .
Abstract of Japanese Patent No. JP1262868, Oct. 19, 1989. cited by other .
Abstract of Japanese Patent No. JP2157039, Jun. 15, 1990. cited by other .
Abstract of Japanese Patent No. JP3195562, Aug. 27, 1991. cited by other .
Abstract of Japanese Patent No. JP4335141, Nov. 24, 1992. cited by other .
Abstract of Japanese Patent No. JP5261246, Oct. 12, 1993. cited by other .
Abstract of Japanese Patent No. JP6285140, Oct. 11, 1994. cited by other .
Abstract of Japanese Patent No. JP63072337, Apr. 2, 1988. cited by other .
Abstract of Japanese Patent No. JP8152409, Jun. 11, 1996. cited by other .
Abstract of SU834073, May 30, 1981. cited by other .
PCT Search Report and Written Opinion for PCT/US2004/011596, Aug. 30, 2004. cited by other .
PCT Search Report and Written Opinion for PCT/US2004/016933, Nov. 2, 2004. cited by other .
Article--Comparison of Non-Invasive Tests to Detect Helicobacter Pylori Infection in Children and Adolescents: Results of a Multicenter European Study, Francis Megraud, The Journal of Pediatrics, vol. 145, No. 2, Feb. 2005, pp. 198-203. cited by other .
Article --Increase of acetone and ammonia in urine headspace and breath during ovulation quantified using selected ion flow tube mass spectrometry, Diskin et al., Physiological Measurement, vol. 24, Jan. 20, 2003, pp. 191-199. cited by other .
Article--Adsorption of Dyes on Nanosize Modified Silica Particles, Guangwei Wu, Athanasia Koliadima, Yie-Shein Her, and Egon Matijevic, Journal of Colloid and Interface Sciences, vol. 195, 1997, pp. 222- 228. cited by other .
Article--Adsorption of Proteins and Antibiotics on Porous Alumina Membranes, Yi Hua Ma, Aseem Bansal, and William M. Clark, Fundamentals of Adsorption, vol. 80, 1992, pp. 389-396. cited by other .
Product Information Sheet for Snowtex.RTM., 6 pages. cited by other .
Article--Significance of Ammonia in the Genesis of Gastric Epithelial Lesions Induced by Helicobacter pylori: An in vitro Study with Different Bacterial Strains and Urea Concentrations, P. Sommi, V. Ricci, R. Fiocca, M. Romano, K.J. Ivey, E. Cova, E. Solcia, and U. Ventura, Digestion, vol. 57, 1996, pp. 299-304. cited by other .
Article--Ammonia vapour in the mouth as a diagnostic marker for Helicobacter pylori infection: preliminary "proof of principle" pharmacological investigations, C. D. R. Dunn, M. Black, D. C. Cowell, C. Penault, N. M. Ratcliffe, R. Spence, and C. Teare, British Journal of Biomedical Science, vol. 58, 2001, pp. 66-76. cited by other .
Article--Purification and Characterization of Urease from Helicobacter pylori, Bruce E. Dunn, Gail P. Campbell, Guillermo I. Perez- Perez, and Martin J. Blaser, The Journal of Biological Chemistry, vol. 265, No. 16, Jun. 5, 1990, pp. 9464-1990. cited by other .
Article--Validation of.sup.13 C-Urea Breath Test for the Diagnosis of Helicobacter Pylori Infection in the Singapore Population, T. S. Chua, K. M. Fock, E. K. Teo, T. M. Ng, Singapore Medical Journal, vol. 43, No. 8, 2002, pp. 408-411. cited by other .
Article--Significance of ammonia produced by Helicobacter pylori, Shigeji Ito, Yoshihiro Kohli, Takuji Kato, Yoshimichi Abe, and Takashi Ueda, European Journal of Gastroenterology & Hepatology, vol. 6, No. 2, 1994, pp. 167-174. cited by other .
Article--Spectrophotometric Assay of Thiols, Peter C. Jocelyn, Methods in Enzymology, vol. 142, 1987, pp. 44-67. cited by other .
Abstract of Japanese Patent No. 7256025, Oct. 9, 1995. cited by other .
PCT Search Report for PCT/US03/32846, Jun. 7, 2004. cited by other .
Derwent Abstract, JP 5106199A, Apr. 1993, Nakajima et al. cited by other .
Derwent Abstract, JP 9143872A, Jun. 1997, Sogawa. cited by other .
Brunauer, S. et al., "Adsorption of Gases in Multimolecular Layers", Journal of American Chemical Society, vol. 60, Feb. 1938, pp. 309-319. cited by other .
Bene, A. et al., "Applicability of a SPME Method for the Rapid Determination of VOCs", Chimia, 56, No. 6, 2002, ISSN 0009-4293, pp. 289-291. cited by other .
Malik, D.J. et al., "Characterisation of Novel Modified Active Carbons and Marine Algal Biomass for the Selective Adsorption of Lead", Water Research, 36, 2002, pp. 1527-1538. cited by other .
Cost, F., Pocket Guide to Digital Printing, Delmar Publishers, Albany, NY, ISBN 0-8273-7592-1, pp. 144-145. cited by other .
Noller, C.R., "Saponins and Sapogenins. VIII. Surface Films of Echinocystic Acid and Derivatives", The Journal of the American Chemical Society, vol. 60, 1938, 3 pages. cited by other .
Antonietti, M., "Synthesis of porous Silica with help from cyclodextrin aggregates", Max-Planck-Institut for Kolloid-und, Germany, 1 page. cited by other .
Maldotti, A. et al., "Immobilization of (n-Bu.sub.4N).sub.4W.sub.10 O.sub.32 on Mesoporous MCM-41 and Amorphous Silicas for Photocatalytic Oxidation of Cycloalkanes with Molecular Oxygen", Journal of Catalysis, vol. 209, 2002, pp. 210-216. cited by other .
Zhang, Q. et al., "Fe-MCM-41 for Selective Epoxidation of Styrene with Hydrogen Peroxide", The Chemical Society of Japan, Chemistry Letters 2001, pp. 946-947. cited by other .
Melde, B.J. et al., "Mesoporous Sieves with Unified Hybrid Inorganic/Organic Frameworks", Chem. Mater., vol. 11, No. 11, 1999, pp. 3302-3308. cited by other .
Polarz, S. et al., "From Cyclodextrin Assemblies to Porous Materials by Silica Templating", Angew. Chem. Int. Ed., vol. 40, No. 23, 2001, pp. 4417-4421. cited by other .
Shi, D. et al., "Uniform Deposition of Ultrathin Polymer Films on the Surfaces of A1.sub.2O.sub.3 Nanoparticles by a Plasma Treatment", University of Cincinnati and University of Michigan, Jun. 2000, pp. 1-15. cited by other .
Santra, S. et al., "Development of novel dye-doped silica nanoparticles for biomarker application", Journal of Biomedical Optics, vol. 6, No. 2, Apr. 2001, pp. 160-166. cited by other .
Buchhammer, M. et al., "Nanoparticles based on polyelectrolyte complexes: effect of structure and net charge on the sorption capacility for solved organic molecules", Colloid Polym. Sci., vol. 278, 2000, pp. 841-847. cited by other .
Brunauer, S. et al., "Adsorption of Gases in Multimolecular Layers", The Journal of the American Chemical Society, vol. 60, Feb. 1938, pp. 309-319. cited by other .
Schaber, P.M. et al., "Study of the urea thermal decomposition (pyrolsis) reaction and importance to cyanuric acid production", American Laboratory, Aug. 1999, pp. 13-21. cited by other .
Bergna, H.E., Editor, "Silanol Groups, Siloxane Bridges, and Physically Adsorbed Water", The Colloid Chemistry of Silica, American Chemical Society 200.sub.th National Meeting, Aug. 26-31, 1990, pp. 22-23 and pp. 52-59. cited by other .
Schweigert, I.V. et al., "Structure and properties of silica nanoclusters at high temperatures", The American Physical Society, Physical Review B, vol. 65, No. 235410, pp. 1-9. cited by other .
Biermann, C.J. et al., Grafting of Poly(ethylenimine) onto Mesylated Cellulose Acetate, Poly(methyl methacrylate) and Poly(vinyl chloride), Carbohydrate Polymers, vol. 12, 1990, pp. 323-327. cited by other .
Yurieva, T.M. et al., Abstract of "Non-hydrothermal synthesis of copper-, zinc- and copper-zinc hydrosilicates", Materials Research Innovations, vol. 5, No. 1, Jun. 2001, 2 pages. cited by other.

Primary Examiner: Mondesi; Robert B
Assistant Examiner: Portner; Ginny
Attorney, Agent or Firm: Dority & Manning, P.A.

Claims

What is claimed is:

1. A kit for detecting Hellcobacter pylori, the kit comprising a breath testing device that contains a substrate on which a visual indicating agent is disposed that is color sensitive to ammonia at a concentration of about 20 to about 500 parts per million, wherein the substrate is located within a tubular carrier portion of a breath collecting device through which the breath of a user passes, wherein the visual indicating agent contains Michler's hydrol, and wherein the substrate further comprises nanoparticles having an average size of less than about 100 nanometers.

2. The kit of claim 1, wherein the visual indicating agent is sensitive to ammonia at a concentration of about 50 to about 400 parts per million.

3. The kit of claim 1, wherein the breath testing device comprises a reference zone.

4. The kit of claim 1, wherein the nanoparticles have a surface area of from about 50 to about 1000 square meters per gram.

5. The kit of claim 1, wherein the nanoparticles include silica, alumina, or combinations thereof.

6. The kit of claim 1, wherein the substrate contains a fibrous material.

7. The kit of claim 6, wherein the fibrous material contains cellulosic fibers.

8. The kit of claim 1, wherein the carrier portion is open at least one end.

9. The kit of claim 8, wherein the carrier portion is substantially flattened.

10. The kit of claim 8, wherein the carrier portion is connected to a balloon.

11. The kit of claim 1, wherein the substrate covers an end of a carrier portion of the breath collecting device.

12. The kit of claim 1, wherein the visual indicating agent is applied to the substrate as a solution.

13. The kit of claim 12, wherein the concentration of the visual indicating agent is from about 0.001 to about 15% wt/wt of the solution.

14. The kit of claim 12, wherein the concentration of the visual indicating agent is from about 0.005 to about 2% wt/wt of the solution.

15. The kit of claim 1, further comprising a source of urea, the urea being hydrolyzable in the presence of a urease enzyme to generate ammonia.

16. The kit of claim 1, wherein the carrier portion is transparent.

Description

BACKGROUND OF THE INVENTION

The present invention relates to a method and device for detecting ammonia odors and uses thereof, in particular for diagnosing helicobacter pylori urease infection.

It is estimated that almost 1 in 10 adults will develop a stomach ulcer at some time in their lives. The bacterium Helicobacter pylori (HP) is known to be the cause of most stomach ulcers and ulcers of the duodenum and, once detected, can be cured by treatment with antibiotics. H. pylori (HP) produces an active form of the urease enzyme, which hydrolyzes urea into ammonia and carbon dioxide. Since ammonia is the key component generated by urease-catalyzed hydrolysis of urea, it follows that the presence of this compound in a person's breath can be used to diagnose helicobacter pylori urease (HPU) infection.

However, there are not many systems to detect ammonia odors, and of those available, most require the use of expensive instrumentation and are complex in operation (and hence not suitable for use by untrained users).

In general, HPU infection is diagnosed by analyzing the difference of exhaled ammonia and/or CO.sub.2 levels from a patient before and after swallowing a dose of radioactive urea. The levels of ammonia in HPU infected persons are usually between 100 to 200 parts per million (ppm) after swallowing the urea, whereas the levels of ammonia in an uninfected person after having swallowed the urea are much the same as before taking the urea (<2 ppm).

The patient first drinks a solution of citric acid as a background test and two baseline expired breath samples are collected in Mylar breath collection balloons from Kimberly-Clark/Ballard Medical Devices of Draper, Utah. The patient then drinks a second solution containing citric acid and carbon-14 labeled urea, and another two expired breath samples are taken after 30 minutes. The samples of expired breath are then analyzed for carbon-14 labeled carbon dioxide. The test is considered positive for H. Pylori infection when the difference between the control breath and the labeled breath samples are greater than 3.5 parts per thousand. This test is both expensive and time consuming.

Therefore, there is a clear need for a simple, safe device which needs neither expensive instrument nor radioactive materials for detecting ammonia from HPU infection and other sources.

SUMMARY OF THE INVENTION

This invention describes a simple device for detecting ammonia odors using a visual indicating agent which changes color when ammonia is present in the breath of a user, in particular when the ammonia is present in the range of 20 to 500 parts per million (ppm), more preferably in the range of 50 to 400 ppm, and most preferably in the range of 75 to 300 ppm.

The indicating agent is also suitable for diagnosing helicobacter pylori urease (HPU) infection, which causes ammonia to be present in a patient with the infection. The development of potential stomach ulcers can thus be inhibited or the stomach ulcers can be treated at an early stage.

The visual indicating agent is typically a dye which is color sensitive to ammonia odors, such as 4,4'-bis(dimethylamino)-benzhydrol (BDMB or Michler's hydrol (MH)), a dye having a similar chemical structure to MH, a triamino-triphenyl-methanol dye such as pararosaniline base (PAB), alpha-naphtholbenzein or any other dye which has high sensitivity for ammonia. The dye may change color by fading to a lighter color, by deepening in color or by actually changing from one color to another.

The device includes a simple carrier portion defining a passage, such as a substantially transparent tube or straw, containing the visual indicating agent. While the invention will be described hereinafter with particular reference to straws and tubes, it will be understood that the invention is useful with various other shapes as well. For example, the shape of the passageway may be cylindrical, triangular, square, almond-shaped and so forth.

The visual indicating agent may be in the form of a powder, in solution (for example, in water, an aqueous solution, toluene or alcohol), or may be coated onto a substrate, such as cellulose tissue or paper, a nonwoven fabric substrate, a woven substrate, glass fiber, cotton, silk, rayon and so forth. The solution may be applied to the substrate and dried so that a dry residue of the indicating agent remains on the substrate. The straw may be substantially flattened to allow for easier storage, while still permitting a patient to blow through the passage in the tube. The device may include a zone with one or more reference colors to allow the user to compare the color of the indicating agent after exposure to his or her breath with the reference color, and so easily determine if there has been a color change.

In one embodiment, the visual indicating agent is coated onto a cellulose substrate which is then inserted into a straw. In another embodiment, the visual indicating agent is again coated onto a cellulose susbstrate, but is placed over one end of a straw.

In both embodiments, when the patient blows into the tube, their breath will pass through the tube and over or through the substrate, thus causing the indicating agent to change color if levels of ammonia which are indicative of HPU infection (generally between 20 and 500 ppm) are present in the patient's breath.

The breath testing devices may be single-use devices which are disposable.

Thus, HPU infection can be detected by a simple color change, and there is no need for radioactive urea and complex instrumentation. No similar system has yet been developed or reported in the literature.

The straw may be connected to the inlet of a breath collection balloon such as the type sold commercially by Kimberly-Clark/Ballard Medical Devices of Draper, Utah for use in the existing H. pylori detection kits (PYtest.TM. 14C-Urea Breath Test). The use of such a balloon would help ensure that the correct amount of breath was sampled, as the patient would simply blow through the straw into the balloon until it was fully inflated.

The patient may ingest urea prior to his or her breath being tested so as to boost the ammonia levels which are detected. After a period of time sufficient to allow HPU, if present, to hydrolyze the urea into ammonia and carbon dioxide, the patient would then blow into a breath testing device. If the patient were infected with HPU, sufficient amounts of ammonia would be present in his or her breath to be detected by the device, and the indicating agent would show a change in color.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a standard curve for the detection of ammonia by Michler's Hydrol-dye;

FIG. 2 shows a standard curve for the detection of ammonia by pararosaniline base (PAB);

FIG. 3 shows the results of a test to monitor the hydrolysis of urea to ammonia and carbon dioxide using PAB;

FIG. 4 shows simple breath testing device according to a first embodiment of the invention in an unassembled state, demonstrating a color change in the indicating agent before (left) and after (right) exposure to ammonia odors;

FIG. 5 shows a second embodiment of a breath testing device according to the invention with a color reference, before (right) and after (left) exposure to ammonia odors;

FIG. 6 shows a third embodiment of the breath testing device according to the invention;

FIG. 7 shows a fourth embodiment of the breath testing device according to the invention;

FIG. 8 shows the breath testing device of FIG. 4 in conjunction with a breath collection balloon.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides simple breath testing devices which are able to detect levels of ammonia odors in a patient's breath which are consistent with helicobacter pylori urease infection without requiring the patient to ingest radio-active chemicals. Thus, the breath testing devices include a visual indicating agent which changes color in response to the ammonia odors, preferably when the ammonia is in the range of 20 to 500 parts per million (ppm), more preferably in the range of 50 to 400 ppm, and most preferably in the range of 75 to 300 ppm.

In one embodiment, the visual indicating agent is 4,4'-bis(dimethylamino)-benzhydrol, also known as "BDMD", "Michler's hydrol" or "MH". This indicating agent is sensitive to both sulphur-containing and amine-containing odors, changing from blue to colorless in the presence of these odors. Michler's Hydrol reacts with amine or sulfur compounds according to the following reaction:

##STR00001##

Michler--s hydrol (MH) and related indicating agents, which are also suitable for use as indicating agents for the present invention, can be represented by the following general formula (I) or (II):

TABLE-US-00001 ##STR00002## Indicating Agent R R' R'' Indicating Agent for Michler's Hydrol (CH.sub.3).sub.2NC.sub.6H.sub.5-- (CH.sub.3).sub.2NC.sub.- 6H.sub.5-- H Ammonia, Amines, (MH) Diamines and Polyamines Pararosaniline (NH.sub.2)C.sub.6H.sub.5-- (NH.sub.2)C.sub.6H.sub.5-- (NH.s- ub.2)C.sub.6H.sub.5-- Ammonia, Amines, Base (PAB) Diamines and Polyamines Alpha- naphtholbenzein (ANB) C.sub.6H.sub.5-- ##STR00003## ##STR00004## Ammonia, Amines, Diamines and Polyamines Naphthochrome Green (NCG) C.sub.6H.sub.5-- ##STR00005## ##STR00006## Ammonia, Amines, Diamines and Polyamines

The dye may change color by fading to a lighter color, by deepening in color or by actually changing from one color to another. Thus, MH changes from blue to colorless, PAB changes from red to colorless and ANB changes from yellow/orange to grey in the presence of ammonia.

The degree of the color change will depend on the concentration of the indicating agent or the concentration of ammonia in the patient's breath. Therefore, in order to observe a color change in response to ammonia levels in the range of 75 to 300 ppm, the concentration of indicating agent which is used is preferably in the range of from 0.01 to 15% wt/wt, more preferably from 0.05 to 2% wt/wt, and most preferably from 0.1 to 0.5% wt/wt.

In one embodiment, the visual indicating agent is coated onto a cellulose substrate which is then inserted into a straw. In another embodiment, the visual indicating agent is again coated onto a cellulose susbstrate, but is placed over one end of a straw.

The substrate, typically a cellulose tissue, may also be coated with nanoparticles to provide a high surface area coating on the substrate, i.e., higher than the cellulose fiber by itself. Thus, the cellulose tissue may be given a boost in surface area by coating it with nanoparticles. The treated substrate may be then coated with the visual indicating dye. It's believed that this high surface area coating helps spread the dye over the silica surface to provide a thinner coating and thus improving the sensitivity of the device.

The average size of the nanoparticles is generally less than about 100 nanometers, in fact it may be from about 1 to about 50 nanometers, and from about 4 to about 20 nanometers. As used herein, the average size of a particle refers to its average length, width, height, and/or diameter.

The nanoparticles may have a surface area of from about 50 square meters per gram (m.sup.2/g) to about 1000 m.sup.2/g, in some cases from about 100 m.sup.2/g to about 600 m.sup.2/g, and in some cases, from about 180 m.sup.2/g to about 240 m.sup.2/g.

In addition, the nanoparticles may also be relatively nonporous or solid. That is, the nanoparticles may have a pore volume that is less than about 0.5 milliliters per gram (ml/g), less than about 0.4 ml/gram, less than about 0.3 ml/g, and even from about 0.2 ml/g to about 0.3 mug. It is believed that the solid nature, i.e., low pore volume, of the nanoparticles may enhance the uniformity and stability of the nanoparticles.

Examples of commercially available alumina nanoparticles include, for instance, Aluminasol.RTM. 100, Aluminasol.RTM. 200 and Aluminasol.RTM. 520, which are available from Nissan Chemical America Corporation of Houston, Tex., USA. Alternatively, silica nanoparticles may be utilized, such as Snowtex-C.RTM., Snowtex-O.RTM., Snowtex-PS.RTM. and Snowtex-OXS.RTM. nanoparticles, which are also available from Nissan Chemical.

Snowtex-OXS.RTM. nanoparticles, for instance, have a particle size of from 4 to 6 nanometers, and may be ground into a powder having a surface area of approximately 509 square meters per gram. Also, alumina-coated silica particles may be used, such as Snowtex-AK.RTM. nanoparticles available from Nissan Chemical.

The breath testing device includes a simple supporting member, such as a substantially transparent tube or straw containing the visual indicating agent. The visual indicating agent may be in the form of a powder, in solution, or may be coated onto a substrate, such as cellulose tissue or paper, cotton, a non-woven fabric substrate, a woven substrate, glass fiber, silk, rayon and so forth. The indicating agent may also be printed onto the substrate, for example by using an inkjet printer.

The straw or tube may be substantially flattened to allow for easier storage, while still permitting a patient to blow through the passage in the tube.

The device may include a zone with one or more reference colors or shades of color to allow the user to compare the color of the indicating agent after exposure to his or her breath with the reference color, and so easily determine if there has been a color change.

In both embodiments, when the patient blows into the tube, their breath will pass through the tube and over or through the substrate, thus causing the indicating agent to change color if levels of ammonia which are indicative of HPU infection (generally from 20 to 500 ppm, more particularly from 50 to 400 ppm and most particularly between 75 to 300 ppm) are present in the patient's breath.

The straw or tube may be connected to the inlet of a breath collection balloon such as the type sold commercially by Kimberly-Clark/Ballard Medical Devices of Draper, Utah for use in the existing H. pylori detection kits (PYtest.TM. 14C-Urea Breath Test). Such a test balloon has a volume of about 0.5 to 2 liters or about 1 lung-full of breath for most individuals. The use of such a balloon helps ensure that the correct amount of breath is sampled. If too much breath is sampled, the device may provide a false positive diagnosis, and if too little breath is sampled, the device may provide a false negative diagnosis. Thus, the patient would simply blow through the straw into the balloon until it was fully inflated.

According to another embodiment, the breath testing devices described above were made smaller in size so that several of the breath testing devices could be easily packaged together in a pocket-sized container. For example, a straw was cut to about 4 cm in length, and a dye-treated substrate was either inserted into the straw or was placed over one end of the straw. The straw was then substantially flattened by laminating it in a standard business card heated laminator so that air was still able to pass through the tube of the straw.

The patient may ingest urea prior to his or her breath being tested so as to boost the ammonia levels which are detected. After a period of time sufficient to allow HPU, if present, to hydrolyze the urea into ammonia and carbon dioxide, the patient would then blow into the breath testing device. If the patient were infected with HPU, sufficient amounts of ammonia would be present in his or her breath to be detected by the device, and the indicating agent would show a change in color.

Unless otherwise specified, chemicals and biochemicals were obtained from Sigma-Aldrich of Milwaukee, Wis.

The most widely used color test is called CIELAB and is discussed in Pocket Guide to Digital Printing by F. Cost, Delmar Publishers, Albany, N.Y. ISBN 0-8273-7592-1 at pages 144 and 145. This method defines three variables, L*, a*, and b*, that correspond to three characteristics of perceived color based on the opponent theory of color perception. The three variables have the following meaning:

L*=Lightness, ranging from 0 to 100. Where 0=dark and 100=light,

a*=Red/green axis, ranging approximately from -100 to 100. Positive values are reddish and negative values are greenish.

b*=Yellow/blue axis, Ranging approximately from -100 to 100. Positive values are yellowish and negative values are blueish.

Because CIELAB color space is somewhat uniform, a single number can be calculated that represents the difference between two colors as perceived by a human being. This difference is termed .DELTA.E and is calculated by taking the square root of the sum of the squares of the three differences (.DELTA.L*, .DELTA.a*, and .DELTA.b*) between the two colors. In CIELAB color space, each .DELTA.E unit is roughly a just-noticeable difference between two colors. So that the two colors have a difference of, for example, 4.4, the human eye can perceive the difference in color between the two colors. CIELAB is therefore a good measure for an objective device-independent color specification system that can be used as a reference color space for the purpose of color management and expression of changes in color.

Color intensities (L*a*b* values) herein were measured using a handheld spectrophotometer from Minolta Co. Ltd. of Osaka, Japan (Model # CM2600d). This instrument utilizes the D/8 geometry conforming to CIE No. 15, ISO 7724/1, ASTM E1164 and JIS Z8722-1982 (diffused illumination/8 degree viewing system. The D65 light reflected by the specimen surface at an angle of 8 degrees to the normal of the surface is received by the specimen-measuring optical system.

Absorbance readings were measured using a microplate reader from Dynex Technologies of Chantilly, Va. (Model # MRX).

An acetate buffer containing 40 mM sodium acetate and 4 M guanidine HCl, pH 5.1, was used for preparations of the indicating agents.

Paper towels or KIMWIPES.RTM. tissues from Kimberly-Clark Corporation of Neenah, Wis., were coated with Snowtex-O.RTM. nanoparticles (pH 4.1), available from Nissan Chemical and were used in the examples described herein with Michler's hydrol dye (MH or BDMB) or pararosaniline base (PAB), where dye was added without acetate buffer, unless otherwise indicated.

The invention will now be described in more detail by way of the following non-limiting examples.

EXAMPLE 1

A reaction mixture was placed into each of 8 vials containing 50 .mu.l of ammonia hydroxide solution as an ammonia source (0, 0.01, 0.02, 0.04, 0.08, 0.16 and 0.64% of ammonia hydroxide, respectively) and 150 .mu.l of MH dye (20 .mu.l of 10.0 mg/ml MH in CH.sub.3CN with 5.0 ml of 40 mM sodium acetate and 4M guanidine HCl, pH5.1). After incubation of all the vials at room temperature for less than 4 minutes, a 200 .mu.l portion from each vial was transferred to a microtiter plate well, and the absorbances were measured at 590 nm using a microtiter plate reader (The absorbances can also be measured in the range of 580-615 nm).

As shown in FIG. 1, a standard curve was derived by plotting the absorbance readings against the concentrations (ppb) of ammonia solutions. In FIG. 1, the x-axis is the concentration of ammonia in parts per billion (ppb) from 10 to 400 and the y-axis is the absorbance at 590 nm from 1 to 0.7. The sensitivity of ammonia detection by MH was shown to be very high.

EXAMPLE 2

A similar study was carried out with another dye, pararosaniline base (PAB), which was shown to be sensitive to amine and ammonia odors. In order to generate a standard curve (FIG. 2), a reaction mixture was placed into each of 8 vials containing 50 .mu.l of an ammonia hydroxide solution as an ammonia source (0, 0.01, 0.02, 0.04, 0.08, 0.16 and 0.64% of ammonia hydroxide, respectively) and 150 .mu.l of PAB solution (10 .mu.l of 10 mg/ml PAB stock solution made in CH.sub.3CN with 5.0 ml of 40 mM sodium acetate and 4 M guanidine HCl, pH5.1). 200 .mu.l of each reaction mixture was transferred to a mictotiter plate well and the wells were incubated at room temperature for 4 to 5 min. The absorbances were then read at 550 nm using a microplate reader. PAB was shown to be highly selective for ammonia and amine odors. In FIG. 2, the x-axis is the concentration of ammonia in parts per billion (ppb) from 10 to 400 and the y-axis is the absorbance at 550 nm from 1 to 1.0.

EXAMPLE 3

PAB was then used to see if it was suitable for use in monitoring the reaction in which urease catalyzes urea to ammonia and carbon dioxide by-products (FIG. 3). Into each of two vials (expt. 1 and expt. 2) was placed 1 ml of a reaction mixture containing 100 .mu.l of 10 mM urea, 850 .mu.l of 10 mM PBS, pH7.3, and 50 .mu.l of 10.0 mg/ml urease. Three control vials were prepared, the first control excluding both urea and urease (control 1), the second control excluding urease but containing urea (control 2), and the third control excluding urea but containing urease (control 3). The vials were vortexed and 50 .mu.l from each vial was transferred to a microtiter plate well. PAB solution (10 .mu.l of 10 mg/ml PAB stock solution made in CH.sub.3CN with 5.0 ml of 40 mM sodium acetate and 4 M guanidine HCl, pH5.1) was then added to each well and the absorbance change with time was monitored at 550 nm using a microplate reader. In FIG. 3, the x-axis is time in minutes and the y-axis is the absorbance at 590 nm from 1 to 1.0. As can be seen in FIG. 3, the three controls had relative constant absorbance over time while the two experimental samples had falling absorbance over time. The PAB did not show any interference with buffer, urea, or urease alone, and was shown to be sensitive for ammonia generated by the urease reaction with urea.

EXAMPLE 4

A first embodiment of a device 10 for detecting HPU infection was designed using an ammonia-odor sensitive dye coated on a cellulose substrate (FIG. 4). Accordingly, 1 mg/ml stock solution of MH-dye was applied onto a SCOTT.RTM. paper towel from Scott Paper of Mississauga, ON, Canada, that had been previously coated with a 1 weight percent (dry) solution of SNOWTEX-O.RTM. nanoparticles and allowed to air dry. The dye-coated paper towel was then cut into 2 cm.times.4 cm strips 12 which were rolled up and each strip 12 was inserted into a clear plastic drinking straw 14 from Glad Products Company of Oakland, Calif.

The devices were tested by injecting known concentrations of ammonia hydroxide into the straws to determine their sensitivity to ammonia odors. A color change (from blue 16 to colorless 18) was noticed and was clearly visible in the presence of ammonia odors.

The experiment was repeated using PAB-dye and alpha-naphtholbenzein dye instead of MH-dye. On exposure to ammonia odors, the dye-coated substrates were observed to change from red to colorless and from yellow/orange to grey, respectively.

The color intensities (La*b*) of the indicating dye showed a clear difference between before and after the exposure to ammonia odors (.about.100 ppb). The level of detection of ammonia odor by either MH or PAB (-100 ppb) is far less compared to the physiological level generated from urea hydrolysis by HP urease, V.sub.max, 1,100.+-.200 .mu.mol of urea hydrolyzed/min/mg of protein.

Several volunteers who had already been diagnosed with HPU infection were also given a number of these straws and were asked to blow into them with one lung-full of breath. A color change of the indicating dye on the substrate within the tubes was easily observed with the unaided eye.

EXAMPLE 5

As an alternative to coating a solution of the indicating agent onto a cellulose substrate as described in example 4 above, a solution of the indicating agent was formulated into an inkjet printable ink. Inkjet printing deposits a thin coating of dye on top of the substrate, potentially allowing a more sensitive color coating on the substrate. The Michler's Hydrol dye solution was formulated with inkjet additives shown in Table 1 below.

TABLE-US-00002 TABLE 1 Inkjet formulation INK COMPONENT VOLUME (ml) Water (deionized) 0.85 Ethylene Glycol 3.0 Glycerol 1.5 Polyethylene Glycol (200 MW) 3.0 1,3-Propanediol 1.5 Michler's Hydrol (1.5 mg/ml) in 40 mM sodium acetate 40.1 and 4 M guanidine HCl, pH 5.1 TOTAL 50

The ink solution was loaded into empty Margarita.RTM. cartridges (part no. 0900400-300) obtained from McDermid-Colorspan of Eden Prairie, Minn. and printed using a wide format McDermid-Colorspan printer (Model XII). A strip of the printed Scott.RTM. paper towel was then exposed to ammonia odor and the blue color was observed to decolorize in about 10 seconds (compared to 3-5 minutes taken to observe the color change of a Scott.RTM. paper towel saturated with MH according to one of the previous examples). Higher sensitivity to the odor was thus observed by inkjet printing the indicating agent onto the substrate.

EXAMPLE 6

KIMWIPES.RTM. tissues were coated with a 5% Snowtex-O.RTM. nanoparticle solution from Nissan Chemical and then air-dried. 5.0 mg/ml stock solution of MH-dye in acetonitrile was applied to the Snowtex-O nanoparticle-coated KIMWIPES.RTM. tissues and a blue color was observed to develop as the applied dye solution dried.

As shown in FIG. 5, a drinking straw 20 from Glad Products Company of Oakland, Calif., was placed on a cardboard strip 22, and a piece of the dye-nanoparticle coated tissue 24 was placed over a first end 25 of the straw. Thus, when a patient breathes into the second end 26 of the straw, their breath would pass through the tissue 24 at the first end of the straw. The straw was placed between two sheets of polyethylene 28 which were then heat-sealed so that only the second end 26 of the straw was exposed. A reference (or control) color strip 30 was also placed between the polyethylene sheets near, but separate from, the first end 25 of the straw and sealed when the polyethylene sheets were heat sealed. This reference strip consists of a piece of the dye-coated tissue and allows the patient to compare the color of the strip 24 at the end of the drinking straw with the reference strip 30 to see if there has been a color change (left), and hence to determine whether he or she has HPU infection. The reference strip could also consist of a scale of two or more shades of blue so that the user could compare the change of color of the tissue in the straw with the scale, and so determine the degree of infection. For example, a slight change in the color could represent mild infection, a more pronounced fade to a lighter blue could represent medium infection, and a complete change from blue to colorless could represent severe infection.

EXAMPLE 7

The devices described in examples 4 and 6 were reduced in size to make them more discreet and easy to carry around.

Two minitiarized breath testing devices were therefore developed:

a) The first prototype (FIG. 7) was prepared by taking the article of example 4 (i.e. a drinking straw 40 into which a dye-treated tissue 42 had been inserted) and making the tube of the drinking straw shorter, for example, 4 cm long. The straw was then placed into a standard business card heated laminator (from Kinko's of Dallas, Tex.) so that the tube was flattened but air was still able to pass through the tube. b) The second prototype (FIG. 6) was prepared by taking the article of example 6 (i.e. a drinking straw 44 having a dye-treated tissue strip 46 covering one end of the straw), and again cutting it to a length of approximately 4 cm. The straw was laminated as above.

EXAMPLE 8

In order to ensure that the HPU testing devices as described above would detect the levels of ammonia which are exhaled by patients having ulcers, it is preferable that a uniform amount of breath is sampled. Thus, the device described in example 4 was attached to a breath collection balloon 50 from Kimberly-Clark/Ballard Medical Devices of Draper, Utah (FIG. 8), discussed above. The patient would therefore blow through the device 10 into the balloon until the balloon becomes inflated, and if the substrate 12 with the indicating agent within the device 10 changed color after exposure to this amount of breath, this would indicate that the patient is suffering from HPU infection.

From the above examples, it can be seen that it is possible to detect HPU infection using a visual indicating agent which is sensitive to ammonia and/or amine odors, rather than requiring a patient to ingest radioactive materials and to use expensive and complex equipment.

While the invention has been described in detail with respect to specific embodiments thereof, it will be apparent to those skilled in the art that various alterations, modifications and other changes may be made to the invention without departing from the spirit and scope of the present invention. It is therefore intended that the claims cover or encompass all such modifications, alterations and/or changes.

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